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See detailMedical Application: Breath Volatile Compounds in Asthma Patients
Stefanuto, Pierre-Hugues ULiege; Zanella, Delphine ULiege; dejong, Thibaut et al

Scientific conference (2019, June)

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See detailIn vitro characterization of lung inflammation mechanisms
Zanella, Delphine ULiege; Dejong, Thibaut; SCHLEICH, FLorence ULiege et al

Poster (2019, May 13)

Exhaled breath analysis has a high potential for early non-invasive diagnosis of lung conditions. Most of lung diseases involve a certain level of inflammation. The characterization of the ongoing ... [more ▼]

Exhaled breath analysis has a high potential for early non-invasive diagnosis of lung conditions. Most of lung diseases involve a certain level of inflammation. The characterization of the ongoing inflammation processes is crucial to define proper medication. The inflammation processes are associated with oxidative stress, which yields to the conversion of chemical from the membranes (as polyunsaturated fatty acids) into volatile compounds secreted by the lungs1. The understanding of the metabolic pathways involved in volatile markers production could open new therapeutic routes for inflammatory diseases. In this study, the lung inflammation was simulated in vitro. A549 epithelial cells, originally isolated from human alveoli, were cultured with and without oxidative agents (from chemical or biological origins) as part of a discovery study for lung inflammation mechanisms characterization. The cell culture volatile organic compounds (VOCs) were extracted by solid phase micro-extraction (SPME) and thermal desorption (TD) and analyzed by comprehensive two-dimensional gas chromatography hyphenated to time-of-flight mass spectrometers (GC×GC-TOFMS). The complete analytical workflow was optimized using central composite design model. On the processing side, different models were designed to compare the volatile profile of the epithelial cells in different inflammatory conditions. Univariate and multivariate feature selection approaches, i.e. Fisher Ratio and Random Forest, were then used to identify specific inflammatory markers. Based on the output, cross-comparison with metabolic pathways databases (e.g. KEGG) was performed. This work is setting up the basis of a multimodal and biomedical project on lung inflammation characterization. [less ▲]

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See detailPhenotypage de l'asthme par volatilomique de l'haleine
Zanella, Delphine ULiege; Stefanuto, Pierre-Hugues ULiege; SCHLEICH, FLorence ULiege et al

Conference (2019, March 26)

L’asthme est l’une des maladies inflammatoires chroniques les plus répandues dans le monde, touchant 235 millions de personnes. Cela représente un problème majeur de santé publique dû aux coûts élevés ... [more ▼]

L’asthme est l’une des maladies inflammatoires chroniques les plus répandues dans le monde, touchant 235 millions de personnes. Cela représente un problème majeur de santé publique dû aux coûts élevés, estimés à 19,3 milliards d’euros par an, liés au traitement et au diagnostic. L’asthme est caractérisé par différents mécanismes inflammatoires des voies respiratoires. En fonction du profil inflammatoire, le traitement le plus adapté sera différent. C’est pourquoi, les traitements actuels ne sont pas efficaces pour une majorité des patients. Le développement de méthode pouvant caractériser efficacement les différents types d’asthme (éosinophilique, neutrophilque, paucigranulocytique, mixte-granulocytique) permettrait l’établissement de traitement personnalisé. L’utilisation de l’haleine comme outils de diagnostic permettrait l’établissement d’une procédure non-invasive pour le suivi de l’asthme. Dans cette étude, nous avons analysé les composés organiques volatils provenant de l’haleine de 521 patients. Les échantillons ont été collectés dans des sacs Tedlar®. Cette étude a été réalisée en deux phases cliniques. La chromatographie gazeuse couplée à la spectrométrie de masse (GC-MS) a été utilisée lors de la première phase. Sept composés ont été identifiés comme marqueurs potentiels de l’asthme par utilisation d’algorithmes d’apprentissage automatique. La phase de validation a été réalisée en analysant l’haleine de 245 patients par chromatographie gazeuse bidimensionnelle exhaustive couplée à la spectrométrie de masse à haute résolution (GC×GC-HRTOFMS). Cette phase de validation a confirmé l’efficacité de cinq composés. L’utilisation des indices de rétention, de la haute résolution et de l’injection de standards internes ont permis l’identification robuste des composés cibles. Dans les deux phases cliniques, les courbes ROC (receiver operating characteristic) ont été construites pour évaluer la performance de la classification dans un scénario de classification binaire. Chaque cohorte de patients a été divisée en un deux sets : un set d’apprentissage servant à la construction du modèle statistique et un set de validation. La phase clinique de validation a confirmé l’efficacité de cinq composés pour la classification des patients sur base de leur phénotype avec une précision de 70%, 60% et 64%. Ces molécules cibles fonctionnent mieux que les méthodes classiques de diagnostiques basées sur l’analyse sanguine ou du NO. La combinaison des analyses sanguines, de mesure du NO et des molécules volatiles cibles fournit la meilleure performance de classification avec une précision de 76%. Cette étude représente la première étude clinique à grande échelle dans le domaine des méthodes de diagnostic basée sur l’haleine. Elle a permis l’identification de marqueurs permettant le phénotypage des patients asthmatiques. Cette étude démontre le potentiel de l’analyse d’haleine pour le diagnostic clinique. [less ▲]

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See detailDéveloppement et validation d'une méthode de diganostique non-ciblée pour la caractérisation des fluides pulmonaires
Zanella, Delphine ULiege; Stefanuto, Pierre-Hugues ULiege; Dubois, Lena ULiege et al

Poster (2019, March)

L’amélioration constante de l’instrumentation analytique génère un intérêt croissant dans les études métabolomiques. Dans ce contexte de caractérisation de mélanges complexes, la chromatographie gazeuse ... [more ▼]

L’amélioration constante de l’instrumentation analytique génère un intérêt croissant dans les études métabolomiques. Dans ce contexte de caractérisation de mélanges complexes, la chromatographie gazeuse bidimensionnelle (GC×GC) constitue une solution efficace pour obtenir un aperçu complet de la composition des échantillons. Cependant, l’augmentation de la résolution analytique s'accompagne d'une augmentation de la complexité des données générées [1,2]. Pour garantir l'utilisation correcte de l'instrumentation analytique, l’optimisation de la méthode et l’établissement d’un protocole de contrôle qualité (QC) adéquats et clairement définis sont nécessaires. Malheureusement, peu d'études GC×GC non ciblées présentent ces aspects importants et une utilisation abusive des termes validation et semi-quantification est florissante dans les publications. Ces biais conduisent au problème général du manque de reproductibilité des études en science, souligné dans les publications récentes de Nature [3,4]. Pour résoudre ce problème, plusieurs initiatives, telles que l'initiative de normalisation en métabolomique (MSI), ont établi des directives générales pour éviter ce manque de robustesse analytique [5,6].Cette étude présente l'application et l'adaptation des directives MSI pour l'analyse de mélanges volatils à l'aide de la GC×GC-TOFMS. Pour illustrer cette approche, les profils volatils de fluide de lavage bronchoalvéolaire (BALF) ont été analysés. Cette procédure médicale est largement répandue et fournit des informations importantes sur les processus immunologiques, inflammatoires et infectieux des voies respiratoires. Tout d'abord, une solution de QC a été créée en regroupant des échantillons de BALF provenant de différents patients. Cette solution a ensuite été utilisée pour l’optimisation de la méthode, c’est-à-dire l’échantillonnage ainsi que les conditions chromatographiques. Sur base des conditions optimales, la validation a été effectuée. La solution QC a également été utilisée comme base pour la mise en œuvre d'un protocole de contrôle de qualité pour l'analyse de volatils dans les matrices biologiques. Cette étude a pour objectif de fournir des conseils pour l’établissement d’une stratégie analytique robuste pour les analyses non ciblées. References 2. Stefanuto, P. H. et al. Advanced method optimization for volatile aroma profiling of beer using two-dimensional gas chromatography time-of-flight mass spectrometry. J. Chromatogr. A 1507, 45–52 (2017). 3. Monya Baker. Seek out stronger science. Nature 537, 703–704 (2016). 5. Sumner, L. W. et al. Proposed minimum reporting standards for chemical analysis: Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics 3, 211–221 (2007). [less ▲]

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See detailMultimodal chemometric approach for the analysis of human exhaled breath in lung cancer patients by TD-GC ×GC-TOFMS
Pesesse, Romain ULiege; Stefanuto, Pierre-Hugues ULiege; SCHLEICH, FLorence ULiege et al

in Journal of Chromatography. B, Biomedical Applications (2019)

Lung cancer is the deadliest cancer in developed countries. To reduce its mortality rate, it is important to enhance our capability to detect it at earlier stages by developing early diagnostic methods ... [more ▼]

Lung cancer is the deadliest cancer in developed countries. To reduce its mortality rate, it is important to enhance our capability to detect it at earlier stages by developing early diagnostic methods. In that context, the analysis of exhaled breath is an interesting approach because of the simplicity of the medical act and its non-invasiveness. Thermal desorption comprehensive two-dimensional gas chromatography time of flight mass spectrometry (TD-GC×GCTOFMS) has been used to characterize and compare the volatile content of human breath of lung cancer patients and healthy volunteers. On the sampling side, the contaminations induced by the bags membrane and further environmental migration of VOCs during and after the sampling have also been investigated. Over a realistic period of 6 h, the concentration of contaminants inside the bag can increase from 2 to 3 folds based on simulated breath samples. On the data processing side, Fisher ratio (FR) and random forest (RF) approaches were applied and compared in regards to their ability to reduce the data dimensionality and to extract the significant information. Both approaches allow to efficiently smooth the background signal and extract significant features (27 for FR and 17 for RF). Principal component analysis (PCA) was used to evaluate the clustering capacity of the different models. For both approaches, a separation along PC-1 was obtained with a variance score around 35%. The combined model provides a partial separation with a PC-1 score of 52%. This proof-of-concept study further confirms the potential of breath analysis for cancer detection but also underlines the importance of quality control over the full analytical procedure, including the processing of the data. [less ▲]

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See detailBreath Print for Asthma Phenotyping
Zanella, Delphine ULiege; Stefanuto, Pierre-Hugues ULiege; SCHLEICH, FLorence ULiege et al

Scientific conference (2018, November 08)

Asthma is one of the most prevalent chronic disorder worldwide, affecting 235 million people. This represents a serious public health issue associated with high health costs, mainly due to the diagnosis ... [more ▼]

Asthma is one of the most prevalent chronic disorder worldwide, affecting 235 million people. This represents a serious public health issue associated with high health costs, mainly due to the diagnosis and treatment. A European study has estimated the total cost of asthma to 19.3 billion euros/year. Asthma is characterized by an inflammation of the airways, involving several different underlying mechanisms. Current therapies remain ineffective in a large proportion of patients. Therefore, the characterization of the different inflammation phenotypes (i.e. eosinophilic, neutrophilic, paucigranulocytic, mixed-granulocytic asthma) is of great importance to provide personalized treatment. Volatile organic compounds from breath of 245 asthmatic patients covering a range of four different asthma phenotypes were analyzed. The breath samples were collected into Tedlar® bags. Thermal desorption coupled with comprehensive two-dimensional gas chromatography – high resolution time-of-flight mass spectrometry was applied for the analysis. The data were split between training and test (60-40%). Random forest algorithm was used to investigate the ability of exhaled breath VOCs to distinguish between the inflammatory profiles. The random forest algorithm was built on 7 significant features highlighted in a first discovery study. ROC curve were constructed to evaluate the classification performance in pair-classification scenario. The AUROC classifications reached 0.71, 0.68 and 0.70 with 70%, 60% and 64% of accuracy. This first large-scale confirmatory study permitted the discrimination of patients according to their respective phenotypes. The present study shows that breath VOCs analysis is an efficient approach for asthma phenotyping and is going to lead to further development in clinical diagnosis. [less ▲]

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See detailGC×GC-(HR)-TOFMS for Metabolomics and Volatilomics
Focant, Jean-François ULiege; Zanella, Delphine ULiege; Di Giovanni, Nicolas ULiege et al

Conference (2018, June)

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See detailGC×GC-(HR)TOFMS : A KEY PLAYER IN YOUR TOOL BOX FOR MEDICAL APPLICATIONS
Stefanuto, Pierre-Hugues ULiege; Zanella, Delphine ULiege; Dubois, Lena ULiege et al

Scientific conference (2018, May)

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See detailASTHMA PHENOTYPING USING BREATH- GC×GC-HRTOFMS
Zanella, Delphine ULiege; Stefanuto, Pierre-Hugues ULiege; Dubois, Lena ULiege et al

Conference (2018, May)

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See detailStep-down of inhaled corticosteroids in non-eosinophilic asthma: A prospective trial in real life.
Demarche, Sophie ULiege; SCHLEICH, FLorence ULiege; HENKET, Monique ULiege et al

in Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology (2018), 48(5), 525-535

BACKGROUND: While non-eosinophilic asthmatics are usually considered poorly responsive to inhaled corticosteroids (ICSs), studies assessing a step-down of ICS in this specific population are currently ... [more ▼]

BACKGROUND: While non-eosinophilic asthmatics are usually considered poorly responsive to inhaled corticosteroids (ICSs), studies assessing a step-down of ICS in this specific population are currently lacking. OBJECTIVES: To assess the proportion of non-eosinophilic asthmatics in whom ICS may be withdrawn without any clinical degradation and to determine the predictive markers of a failure to stop treatment with ICS. METHODS: This prospective study was completed by 36 non-eosinophilic asthmatics, defined by sputum eosinophils <3% and blood eosinophils <400/muL. In these patients, whichever the baseline asthma control level, the dose of ICS was gradually reduced every 3 months until they met the failure criteria or successfully discontinued ICS for 6 months. The failure criteria were an ACQ score >/=1.5 with an increase from baseline >0.5 or a number of severe exacerbations during the study which was greater than the number during the year prior to the baseline visit. Receiver-operating characteristic (ROC) curves were constructed to assess predictors of a failure to stop ICS. This study is registered with ClinicalTrials.gov, number NCT02169323. RESULTS: In 14 patients (39%), ICSs were completely withdrawn, and in 10 further patients (28%), ICS were stepped-down to a reduced ICS dose without any deterioration of asthma control and exacerbation rate. Baseline predictors of a failure to stop ICS were a greater age (area under ROC curve [ROC AUC] and [95% CI]: 0.77 [0.62-0.93]) and elevated blood eosinophils (ROC AUC [95% CI]: 0.77 [0.61-0.93]). After the first step-down of ICS, the best predictor was an elevated blood eosinophil count (ROC AUC [95% CI]: 0.85 [0.72-0.99]). CONCLUSIONS & CLINICAL RELEVANCE: Withdrawing or reducing the dose of ICS is feasible in two-thirds of non-eosinophilic asthmatics irrespective of baseline asthma control. An elevated blood eosinophil count may predict the failure to stop ICS. [less ▲]

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See detailTentative differentiation of asthma phenotypes using GC×GC-TOFMS
Zanella, Delphine ULiege; Pesesse, R; SCHLEICH, FLorence ULiege et al

Conference (2018, January)

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